EP1446422B1 - Methyliertes rekombinantes mykobakterielles antigen, dass einem heparin-bindenden hämagglutinin (hbha) entspricht - Google Patents

Methyliertes rekombinantes mykobakterielles antigen, dass einem heparin-bindenden hämagglutinin (hbha) entspricht Download PDF

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EP1446422B1
EP1446422B1 EP02795382A EP02795382A EP1446422B1 EP 1446422 B1 EP1446422 B1 EP 1446422B1 EP 02795382 A EP02795382 A EP 02795382A EP 02795382 A EP02795382 A EP 02795382A EP 1446422 B1 EP1446422 B1 EP 1446422B1
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hbha
recombinant
immunogenic
methylated
heparin
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EP1446422A2 (de
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Kévin PETHE
Franco Menozzi
Camille Locht
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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Institut National de la Sante et de la Recherche Medicale INSERM
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention is part of the research and development of new vaccines for the treatment of mycobacterial infections, including tuberculosis.
  • the present invention relates to a methylated immunogenic recombinant peptide sequence corresponding to the heparin-binding hemagglutinin (HBHA) identified in mycobacterial strains such as Mycobacterium tuberculosis and M. bovis. BCG ( Menozzi et al. 1996. J. Exp. Med. 184: 993-1001 ).
  • HBHA heparin-binding hemagglutinin
  • the invention also relates to methods for preparing an immunogenic peptide sequence comprising recombinant HBHA, said sequence being methylated by post-translational modification.
  • the invention particularly relates to methods of chemical or enzymatic methylation of a peptide sequence comprising HBHA and previously produced in unmethylated recombinant form.
  • the invention further relates to recombinant tools, vectors and recombinant cell hosts for carrying out the chemical or enzymatic methods of post-translational methylation of recombinant HBHA.
  • the invention further relates to immunogenic compositions comprising native, recombinant or methylated HBHA, such compositions being useful for the preparation of vaccines against mycobacterial infections.
  • Mycobacteria are bacilli whose habitat is very diverse. Depending on the species, these bacteria can colonize soil, water, plants, animals and / or humans. Some species, such as M. smegmatis, are non-pathogenic saprophytes. Other species, in However, they are more or less severe pathogens for animals and / or humans. Thus, M. avium causes infections in the bird. M. bovis is responsible for bovine tuberculosis, although it has also been implicated in cases of human tuberculosis. In humans, tuberculosis is mainly caused by the highly pathogenic species M. tuberculosis. M. leprae is responsible for leprosy, another human pathology that is mainly prevalent in developing countries.
  • Tuberculosis remains a serious public health problem at the moment, as it is the leading cause of death from a single infectious agent.
  • the World Health Organization (WHO) reported 8.8 million cases of tuberculosis in 1995 ( Dolin et al. 1994. Bull. WHO 72: 213-220 ). More recently, WHO has published alarming figures, with 10 million new TB cases annually and 3 million deaths a year ( Dye et al. 1999. J. Am. Med. Assoc. 282: 677-686 ). It is estimated that one-third of the world's population is infected with M. tuberculosis. However, not all infected people develop the disease.
  • M. tuberculosis infections were effectively controlled by certain antibiotics, including rifampicin, isoniazid and pyrazinamide.
  • antibiotic therapy quickly showed limitations in the curative treatment of tuberculosis because, on the one hand, the emergence of antibiotic-resistant strains of M. tuberculosis , in particular isoniazid, and on the other hand, the toxicity of certain anti-tuberculosis molecules, including pyrazinamide.
  • BCG vaccine Bacillus Calmette and Guérin
  • This vaccine consists of a live form of a strain of M. bovis, isolated in 1908 from a cow, and made avirulent in vitro to allow parenteral administration in humans.
  • this vaccine is currently controversial in that it has limitations, particularly in terms of effectiveness. Indeed, according to numerous clinical trials carried out by the monad, the protective efficacy obtained with the BCG vaccine varies from 0 to 85% ( Fine, PE 1989. Rev. Infect. Dis. 11 Suppl. 2: S353-S359 ).
  • the present invention therefore aims to overcome the disadvantages of BCG vaccine, by proposing a new immunogenic composition that can be used as a vaccine against tuberculosis.
  • This immunogenic composition can also be used more generally, in the context of the prevention of mycobacterial infections.
  • Tuberculosis is a contact disease that is transmitted by air. Once inhaled, the germs of M. tuberculosis reach the lungs, which are the initial source of infection. From the lungs, the germs are rapidly disseminated through the blood or lymphatic system to other areas of the body.
  • M. tuberculosis One of the major events in the pathogenesis of tuberculosis is the adherence of microorganisms to target cells. Alveolar macrophages have long been considered the gateway for M. tuberculosis, and are thought to transport bacteria from the lungs to other organs. However, it has recently been shown that M. tuberculosis is also able to interact with epithelial cells, including M cells, which could allow bacilli to cross directly the epithelial barrier ( Teitelbaum et al. 1999. Immunity 10: 641-650 ). The relative contribution of each of these mechanisms, as well as the bacterial factors involved in extra-pulmonary dissemination of M. tuberculosis remain unknown.
  • HBHA heparin-binding hemagglutinin adhesin
  • HBHA does not play a leading role in the initial stages of TB infection, nor in the persistence of mycobacteria in the lungs ( Pethe et al. 2001. Nature 412: 190-194 ). These researchers also showed that HBHA was not required for colonization and survival in the spleen. In contrast, HBHA plays a crucial role in extra-pulmonary dissemination of mycobacteria. This adhesin is therefore a virulence factor whose binding to non-phagocytic cells represents an essential step in the process of disseminating mycobacteria from the lungs to the spleen and potentially other organs such as liver, bones, kidneys or possibly the brain.
  • the object of the present invention is to use, as part of an essentially prophylactic treatment, the antigenicity of HBHA, whose role is essential in the dissemination of microorganisms in infected individuals.
  • the inventors disclose the nature of the post-translational modification carried by the native HBHA, namely a complex covalent methylation, said modification conferring on it a protective antigenic power against mycobacterial infections.
  • the invention therefore relates to an immunogenic recombinant peptide sequence comprising a methylated antigen corresponding to the native HBHA or the C-terminal part thereof.
  • protein within the meaning of the present invention, all or part of the peptide sequence of HBHA, provided however to include at least the C-terminal domain of said HBHA.
  • sequence considered comprises at most the C-terminal domain of the HBHA
  • peptide is advantageously used.
  • peptides denotes products resulting from an enzymatic digestion of HBHA.
  • use of the term “peptide” is not limited to this case, “peptide” may also be synonymous with “protein” in the context of the invention.
  • a “recombinant" peptide sequence according to the invention corresponds to a peptide sequence obtained by expression, in a heterologous cellular host, of a nucleotide sequence coding for said peptide sequence.
  • said heterologous cell host may be a bacterium that does not belong to the Mycobacterium genus , for example E. coli, or other organisms such as yeasts and animal and plant cells.
  • nucleotide sequence refers to any DNA sequence encoding a peptide sequence as defined in the context of the present invention.
  • an “antigen” refers to any peptide sequence according to the present invention having an immunogenic capacity.
  • an antigen according to the invention may be restricted to the carboxyl-terminal heparin binding domain of HBHA.
  • the immunogenic recombinant peptide sequence according to the present invention is methylated at the level of the heparin binding domain of HBHA.
  • the methyl groups are carried by lysine residues present in said heparin binding domain.
  • the methyl groups are borne by all or only part of the lysine residues present in the C-terminal domain of HBHA, provided that the thus methylated peptide sequence exhibits immunogenic activity.
  • At least thirteen lysine residues out of the fifteen present in the C-terminal domain are methylated.
  • the two unmethylated lysine residues are in particular the distal amino acids of the sequence indicated above of the C-terminal domain of HBHA.
  • the methylated lysine residues are preferably mono- or dimethylated.
  • the recombinant immunogenic peptide sequence according to the present invention namely the recombinant form of the post-translational methylated HBHA, is recognized by the monoclonal antibody 4057D2, and this, unlike the unmethylated recombinant form of said HBHA, as described in the examples that follow.
  • the invention also relates to methods for preparing an immunogenic peptide sequence comprising recombinant HBHA, said sequence being methylated by post-translational modification.
  • the purification step of the HBHA protein can be carried out before or, according to another embodiment, after the step of methylation of said protein.
  • the preparation method according to the invention makes it possible to obtain the methylated recombinant HBHA protein or, alternatively, any methylated peptide comprising at least the heparin binding domain of said protein.
  • said methylated peptide obtained by the method according to the invention corresponds to said heparin binding domain of HBHA.
  • the heterologous cell host used in the context of the preparation process according to the invention is a bacterium, in particular E. coli or M. smegmatis.
  • the host used is M. smegmatis.
  • Protein purification methods are known to those skilled in the art and do not, as such, be the subject of the present invention.
  • the heparin binding properties conferred by the C-terminal domain of HBHA can be exploited by purifying said HBHA by heparin-sepharose affinity (Pethe et al., 2000, supra). .
  • the invention relates to methods of chemical and enzymatic methylation of a peptide sequence comprising HBHA previously produced in unmethylated recombinant form.
  • production in a recombinant form is meant the production of the peptide by expression in a prokaryotic heterologous host or eukaryotic whatever it is.
  • the production can be obtained from a cell culture or in vivo as, for example, in milk or in a plant.
  • the chemical methylation according to the invention is inspired by the literature ( Means, GE 1977. Meth. Enzymol. 47: 469-478 ).
  • the chemical methylation reaction is carried out in a solution comprising formaldehyde and NaBH 4 .
  • the enzymatic methylation methods according to the invention can be carried out using one or more mycobacterial methyltranferases.
  • Said methyltransferases catalyze the transfer of methyl groups from a donor to an acceptor, in this case the peptide sequence of the recombinant HBHA previously purified.
  • the methyl radical donor may be especially S-adenosylmethionine (AdoMet), well known to those skilled in the art.
  • methyltransferase (s) are present and active in total protein extracts of mycobacteria such as M. bovis BCG and M. smegmatis .
  • the mycobacterial methyltransferase (s) can be purified from the total protein extracts of mycobacterial strains before being placed in the reaction medium in order to catalyze the transmethylation reaction (s) from the donor to the acceptor.
  • Recombinant cellular vectors and hosts can be used for the implementation of enzymatic processes for post-translational methylation of recombinant HBHA.
  • a recombinant cell host is capable of coexpressing the nucleotide sequences encoding HBHA and the mycobacterial methyltransferase (s).
  • Said cellular host is preferably a bacterium, and in particular an E. coli strain .
  • coexpress defines, in the context of the invention, the faculty, for a given cellular host, of expressing at least two distinct nucleotide sequences.
  • the cellular host is characterized in that it can simultaneously host at least two recombinant vectors, vectors one of which codes for HBHA whereas the other (s) encode the mycobacterial methyltranferase (s).
  • the cellular host can harbor as many recombinant vectors as different proteins to produce, each vector then encoding a distinct recombinant mycobacterial protein.
  • vector expression vector
  • plasmid plasmid
  • All or some of the recombinant mycobacterial proteins are encoded by the same expression vector.
  • the cellular host may harbor a single expression vector from which all mycobacterial proteins, namely HBHA and the methyltransferase (s), are produced.
  • each mycobacterial protein, HBHA or methyltranferase is controlled by separate regulatory sequences or, in another embodiment, by the same regulatory sequences.
  • An expression vector may advantageously encode HBHA and at least one mycobacterial methyltransferase.
  • an expression vector encodes a single recombinant mycobacterial protein selected from HBHA and the methyltransferase (s).
  • the present invention relates not only to cellular hosts and expression vectors, as defined above, for carrying out the enzymatic methylation methods according to the invention.
  • the invention further relates to methylated immunogenic recombinant peptide sequences obtainable in vivo by an enzymatic method, or in vitro by a chemical or enzymatic process.
  • the invention further relates to immunogenic compositions comprising native, recombinant or methylated HBHA, such compositions being useful for the preparation of vaccines against mycobacterial infections.
  • an immunogenic composition according to the present invention comprises, in a pharmaceutically acceptable formulation, an active ingredient which is a methylated peptide sequence chosen from the peptide sequence of the native HBHA and the peptide sequence of the recombinant HBHA.
  • a "pharmaceutically acceptable formulation” corresponds to a medicinal formulation that can be used in humans, at acceptable doses in vivo with respect to the toxicity and pharmacology of the compounds concerned, while being therapeutically, and in particular from the immunogenic point of view.
  • the methylated peptide sequence serving as the active principle is associated with one or more adjuvants.
  • the term "adjuvant” or “adjuvant compound” means a compound capable of inducing or increasing the specific immune response with respect to an antigen or immunogen, said response being indifferently a humoral and / or cellular response.
  • This immune response generally involves stimulating the synthesis of immunoglobulins specific for a given antigen, in particular IgG, IgA and IgM, or cytokines.
  • the active ingredient, the methylated peptide sequence of HBHA, as well as the adjuvant (s), are generally mixed with pharmaceutically compatible excipients, such as water, a salt buffer, dextrose, glycerol, ethanol, or the like. mixtures thereof.
  • Such immunogenic compositions are prepared in the form of liquid solutions, or injectable suspensions, or else in a solid form, for example freeze-dried, suitable for dissolving prior to injection.
  • An immunogenic composition according to the present invention is formulated to allow administration by routes as diverse as the nasal, oral, subcutaneous, intradermal, intramuscular, vaginal, rectal, ocular, or auricular routes.
  • auxiliary compounds may in particular be wetting agents, emulsifiers or buffers.
  • an immunogenic composition according to the invention comprises, per dose, from 0.1 to 20 ⁇ g, and preferably 5 ⁇ g, of purified HBHA protein.
  • M. bovis BCG 1173P2 WHO
  • M. tuberculosis M. tuberculosis
  • M. smegmatis MT103 MC 2155 The strains of M. bovis BCG 1173P2 (WHO), M. tuberculosis and M. smegmatis MT103 MC 2155 were grown in the middle Sauton (Menozzi et al., 1996, supra).
  • E. coli strain BL21 (DE3) (pET- hbhA ) (Pethe et al., 2000, supra) was cultured in LB medium supplemented with 30 ⁇ g / ml kanamycin.
  • HBHAs Native and recombinant HBHAs were isolated as described (Menozzi et al., 1996, supra, Pethe et al., 2000, supra). The final purification step was carried out by reverse phase HPLC (Beckman Gold System) using a nucleosyl-C18 type column equilibrated in 0.05% trifluoroacetic acid. Elution was carried out by means of a linear gradient from 0 to 80% acetonitrile prepared in 0.05% trifluoroacetic acid.
  • Samples (0.1 to 10 picomoles) were prepared by the "dry drop” method.
  • a volume of solution of 0.5 ⁇ l was mixed with ⁇ -cyano-4-hydroxycinnaminic acid dissolved extemporaneously at the rate of 10 mg / ml in a solution containing 50% of CH 3 CN and 0.1% trifluoroacetic acid. After depositing on the assay plate, the samples were dried. Mass spectrometry analyzes were performed using a MALDI-TOF Voyager-DE-STR device (Applied BioSystems, Foster City, CA). Deposits containing peptides of less than 3000 Da were analyzed using the following adjustment parameters: positive and reflective modes, 20 kV acceleration voltage, 61% gate voltage, 90 ns extraction delay , and lower mass threshold of 500 Da.
  • the adjustment parameters are: positive and reflective modes, 25 kV acceleration voltage, 65% gate voltage, 250 ns extraction delay, and threshold. lower mass of 1000 Da.
  • the spectra were calibrated externally from the monoisotopic ions [M + H + ] of different peptides.
  • the purified native HBHA by HPLC was hydrolysed under constant heating at 110 ° C in 6N HCl solution for 14-16 h.
  • the amino acid composition was determined using a Beckman Gold System analyzer.
  • the amino-terminal peptide sequence was determined by the automated method of Edman degradation using a pulsed liquid device (Procise 492, Applied BioSystems) equipped with a 120A amino acid analyzer. For each sequence determination step, the samples comprised 10 to 20 ⁇ l, which corresponded to a peptide amount ranging from 250 to 500 picomoles.
  • mice were transferred to a P3 confinement.
  • mice were immunized three times at two-week intervals subcutaneously at the base of the tail, with 5 ⁇ g of native HBHA per dose, emulsified or not in a solution of dimethyldioctadecylammonium (DDA, 150 ⁇ l / dose, Sigma ) and monophosphoryl lipid A (MPL, 25 ⁇ g / dose, Sigma).
  • DDA dimethyldioctadecylammonium
  • MPL monophosphoryl lipid A
  • mice were infected intravenously in the lateral vein of the tail by an inoculum of 10 5 CFU of M. tuberculosis suspended in phosphate buffer (PBS, pH 7.4), in a final volume of 200 .mu.l. Four mice per group were sacrificed after six weeks. The number of bacteria was determined in the spleen, liver and lungs of each infected mouse, plating the dilutions of the milled organs on 7H11 medium.
  • PBS phosphate buffer
  • mice vaccinated with BCG were plated on 7H11 dishes containing 2 ⁇ g / ml of 2-thiophenecarboxylic acid hydrazide, in order to inhibit the growth of residual BCGs. Colonies were counted after two weeks of incubation at 37 ° C. The protective efficacy was expressed in log 10 of reduction of the number of bacteria present in the organs of the immunized mice in comparison with the enumeration relative to the group which had received the adjuvant alone. The results were obtained from groups of four mice.
  • the lymphocytes of the rats were purified as described ( Andersen et al. 1991. Infect. Immun. 59: 1558-1563 ).
  • the lymphocytes of four mice per experiment were cultured in 96-well plates (NUNC) containing 2.10 5 cells / well, in 200 ⁇ l of RPMI 1640 (Gibco, France) supplemented with 50 ⁇ M of 2-mercaptoethanol (Merck, Germany), 50 ⁇ g / ml penicillin-streptomycin (Gibco), 1 mM glutamax (Gibco) and 10% fetal calf serum (Roche).
  • Concanavalin A at 5 ⁇ g / ml was used as a positive control of cell viability.
  • Native HBHA was used at a final concentration of 5 ⁇ g / ml.
  • the supernatants were recovered 72 hours after the start of the stimulation in order to assay the IFN- ⁇ .
  • IFN- ⁇ was detected by a sandwich ELISA.
  • the anti-IFN- ⁇ monoclonal antibodies used were obtained from clones R4-6A2 (Pharmingen, USA) for capture, and SMG1-2 (Pharmingen) for detection.
  • Mass spectrometry analysis revealed that the recombinant HBHA had a molecular weight (MW) of 21340, which corresponded to the PM deduced from the nucleotide sequence coding for the mycobacterial HBHA ( hbhA gene or Rv0475 in M. tuberculosis H37Rv) ( Menozzi et al., 1998, supra).
  • the PM of the native HBHA was 21610, a PM of 270 higher than that of the recombinant HBHA.
  • the mycobacteria-produced HBHA was modified, which was not found in the recombinant protein produced by E. coli.
  • the native and recombinant HBHAs were subjected to hydrolysis by Endo-Glu, and the mass of peptides obtained was determined by mass spectrometry.
  • the only difference between native and recombinant HBHA has been identified at the carboxy-terminal domain of said proteins.
  • the mass of this domain was 4342 Da for native HBHA, and only 4076 Da for recombinant HBHA. This difference of about 270 Da corresponded to the difference in mass measured between whole HBHA proteins.
  • the post-translational modification (s) of the native HBHA could be located in the C-terminal domain.
  • the mass spectrum corresponding to said domain consisted of a single peak for the recombinant HBHA, whereas it had five peaks for the native HBHA, these peaks being separated from each other by 14 Da ( Figure 1 ).
  • the sequence of the heparin binding domain was determined by the Edman degradation method according to standard procedures. This study revealed that only lysines were modified. In addition, of the fifteen lysine residues present in the C-terminal domain of HBHA, only two had the retention time of the lysine standard. The other thirteen residues had retention times corresponding to glutamine and / or arginine standards.
  • Amino acid analysis including mono-, di- and trimethyllysine as standards, confirmed this result.
  • the recombinant HBHA was chemically methylated and then subjected to mass spectrometry analysis. As indicated on the figure 3 , the mass of the peptide corresponding to the C-terminal domain of the HBHA recombinant increased as chemical methylation progressed.
  • the degree of methylation influenced the reactivity of the peptides with monoclonal antibodies 3921 E4 and 4057D2 (Rouse et al., 1991, supra) ( Figure 4 ).
  • the recombinant HBHA was not recognized by the 4057D2 antibody, whereas it was weakly recognized by the 3921 E4 antibody.
  • the degree of methylation of recombinant HBHA affected its affinity for these two antibodies differently, showing that methylation of a protein could play an important role in its antigenicity.
  • a specific in vitro methylation assay of recombinant HBHA was developed from a mycobacterial lysate. Mycobacterial cultures were lysed by sonication. The total lysates, as well as cytoplasmic and parietal fractions, were used as enzyme sources to attempt to transfer moieties [14 C] methyl donor [1 4 C-methyl] AdoMet to the acceptor represented by the recombinant HBHA. Incubation of total lysates of M. tuberculosis, M. bovis BCG and M.
  • the proteins present in a mycobacterial lysate are separated by ion exchange chromatography, HPLC or affinity, following the fractions capable of catalyzing the transmethylation reaction from [ 14 C-methyl] AdoMet on recombinant HBHA. Such enrichments are continued until a sample is obtained in which the methyltransferase (s) are sufficiently pure to determine its sequence.
  • the gene (s) encoding the methyltransferase (s) are identified and then cloned according to the techniques known to the human job.
  • M. smegmatis does not express HBHA (Pethe et al., 2001, supra). However, it was possible to transfer [ 14 C] methyl groups from [ 14 C-methyl] AdoMet onto recombinant HBHA using a lysate of this microorganism ( Figure 2 ). It was therefore suggested that Mr. Smegmatis possessed the enzymatic machinery responsible for the transmethylation reaction of HBHA. In order to verify this hypothesis, M. smegmatis strain MC 2 155 was transformed with a derivative of plasmid pRR3 containing the hbhA gene . (Rv0475) encoding HBHA in M. bovis BCG, to obtain the strain M.
  • the native HBHA is alternately purified from the transformed M. smegmatis strain (pRR-hbhA).
  • the immunization protocol was inspired by the literature ( Brandt et al. 2000. Infect. Immun. 68: 791-795 ). DDA and MPL adjuvants were respectively used at 150 ⁇ g and 25 ⁇ g per dose.
  • Group 1 was vaccinated with the adjuvant alone contained in 200 .mu.l of PBS buffer.
  • Group 2 was vaccinated with 5 ⁇ g of purified native HBHA and emulsified in 200 ⁇ l of PBS-adjuvant mixture.
  • Group 3 was vaccinated with 5 ⁇ g of native HBHA, dissolved in 200 ⁇ l of PBS. The mice received three injections of the different preparations two weeks apart.
  • a fourth group (positive control) was vaccinated with a dose of 5.10 5 CFU of BCG.
  • mice vaccinated with HBHA (groups 2 and 3) produced high levels of IgG1, and also produced IgG2a, IgG2b, as well as IgG3.
  • These types of antibodies reflected the generation of a mixed TH1 / TH2 response.
  • the presence of the adjuvant (group 2) did not change the profile of the response to the HBHA protein alone (group 3). However, said adjuvant made it possible to produce approximately 10 times more different IgGs (Table 1).
  • mice per group were sacrificed ten weeks after the first injection. Lymphocytes were recovered and stimulated in vitro by native HBHA. After stimulation, IFN-gamma production was tested. As indicated on the figure 5 only lymphocytes purified from group 2 mice, vaccinated with the native adjuvanted HBHA, produced IFN- ⁇ specific for said HBHA.
  • mice were infected intravenously with 10 5 CFU of M. tuberculosis.
  • mice per group were sacrificed six weeks after infection to enumerate the number of CFUs present in the different organs of the mice. Bacterial burden was determined in the liver, spleen and lungs of animals.
  • Resistance was defined as the difference in bacterial load, expressed in log 10 , between control group 1, vaccinated with adjuvant alone, and groups 2 and 4, respectively vaccinated with adjuvanted HBHA and with BCG. Table 2 below indicates the effectiveness of the protection induced by the different immunizations.
  • the enumeration of CFUs has shown that the immune response generated by native HBHA is capable of rendering the mouse partially resistant to M. tuberculosis infection .
  • the resistance observed was, moreover, of the same order of magnitude, whether it be the native HBHA or the reference vaccine of the state of the art, namely BCG.
  • injections of native HBHA protected the mouse from infection with M. tuberculosis, in proportions close to those of the BCG vaccine.
  • recombinant methylated HBHA in that it is immunogenic, gives the animal resistance to infection with M. tuberculosis as effective as that induced by native HBHA.
  • one of the objects of the present invention is a subunit vaccine for the treatment of mycobacterial infections and comprising, in its formulation, the native HBHA.
  • a preferred object of the invention relates to a subunit vaccine for the treatment of mycobacterial infections advantageously characterized in that it comprises, in its formulation, the recombinantly methylated HBHA, that is to say ie produced by a recombinant cell host meticulously chosen to meet the industrial and sanitary requirements.

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Claims (28)

  1. Immunogenes rekombinantes Peptid, dadurch gekennzeichnet, dass es eine methylierte Form des Expressionsprodukts einer ein mycobaktierielles Antigen vom Typ Heparin-bindendes Hämagglutinin (HBHA) kodierenden Nukleotidsequenz im Bereich der Heparin-Bindungsdomäne des HBHA mit der Sequenz KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQK ist.
  2. Immunogenes rekombinantes Peptid nach Anspruch 1, dadurch gekennzeichnet, dass das mycobakterielle Antigen vom Typ Heparin-bindendes Hämagglutinin mittels M. bovis BCG oder M. tuberculosis erhalten wird.
  3. Immunogenes rekombinantes Peptid nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Nukleotidsequenz für einen Teil des HBHA-Proteins kodiert, das wenigstens die Heparin-Bindungsdomäne des HBHA enthält.
  4. Immunogenes rekombinantes Peptid nach einem beliebigen der Ansprüche 1 bis 3, dadurch gekennzeichnet, dass die Methylgruppen von in der Heparin-Bindungsdomäne des HBHA liegenden Lysin-Resten getragen sind.
  5. Immunogenes rekombinantes Peptid nach Anspruch 4, dadurch gekennzeichnet, dass die Lysin-Reste mono- oder dimethyliert sind.
  6. Immunogenes rekombinantes Peptid nach Anspruch 4 oder 5, dadurch gekennzeichnet, dass die Methylgruppen von allen oder von Teilen der in der Heparin-Bindungsdomäne des HBHA liegenden Lysin-Reste getragen sind.
  7. Immunogenes rekombinantes Peptid nach einem beliebigen der Ansprüche 4 bis 6, dadurch gekennzeichnet, dass die Methylgruppen von allen in der Heparin-Bindungsdomäne des HBHA liegenden Lysin-Resten getragen sind.
  8. Verfahren zum Herstellen eines immunogenen rekombinanten Peptids gemäß einem beliebigen der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass es wenigstens die folgenden Schritte umfasst:
    (a) Produktion des rekombinanten HBHA-Proteins durch einen rekombinanten Zellwirt;
    (b) Reinigung des Proteins; und
    (c) seine posttranslatorische Methylierung;
    wobei die Reihenfolge der beiden letzten Schritte vertauscht werden kann.
  9. Herstellungsverfahren nach Anspruch 8, dadurch gekennzeichnet, dass das rekombinante HBHA-Protein von seiner Heparin-Bindungsdomäne gebildet ist.
  10. Herstellungsverfahren nach Anspruch 8 oder 9, dadurch gekennzeichnet, dass der rekombinante Zellwirt ein Bakterium ist.
  11. Herstellungsverfahren nach Anspruch 10, dadurch gekennzeichnet, dass das Bakterium M. smegmatis ist.
  12. Herstellungsverfahren nach einem beliebigen der Ansprüche 8 bis 11, dadurch gekennzeichnet, dass der Methylierungsschritt chemisch erfolgt.
  13. Herstellungsverfahren nach einem beliebigen der Ansprüche 8 bis 11, dadurch gekennzeichnet, dass der Methylierungsschritt enzymatisch erfolgt.
  14. Herstellungsverfahren nach Anspruch 13, dadurch gekennzeichnet, dass die Methylierungsreaktion durch wenigstens eine in den Extrakten der gesamten Mycobakterienproteine enthaltene Methyltransferase katalysiert wird.
  15. Herstellungsverfahren nach Anspruch 14, dadurch gekennzeichnet, dass wenigstens eine Methyltransferase in den Extrakten der gesamten Proteine von M. bovis BCG oder M. smegmatis enthalten ist.
  16. Methylierte immunogene rekombinante Peptide, die durch ein Verfahren gemäß einem beliebigen der Ansprüche 8 bis 15 erhältlich ist.
  17. Verwendung einer methylierten Form von nativem HBHA oder einer methylierten Form von rekombinantem HBHA im Bereich der Heparin-Bindungsdomäne mit der Sequenz KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQK zur Gewinnung von Impfstoffen gegen mycobakterielle Infektionen.
  18. Verwendung nach Anspruch 17 zur Gewinnung von Impfstoffen gegen Infektionen mit M. bovis oder M. tuberculosis.
  19. Verwendung nach Anspruch 17 oder 18, dadurch gekennzeichnet, dass die rekombinante Form ein immunogenes rekombinantes Peptid gemäß einem beliebigen der Ansprüche 1 bis 7 ist.
  20. Immunogene Zusammensetzung, dadurch gekennzeichnet, dass sie als Wirkstoff eine methylierte Form des nativen HBHA oder ein methylierte Form des rekombinanten HBHA im Bereich der Heparin-Bindungsdomäne mit der Sequenz KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQK und wenigstens einen pharmazeutisch kompatiblen Trägerstoff umfasst.
  21. Immunogene Zusammensetzung nach Anspruch 20, dadurch gekennzeichnet, dass die methylierte Form mit einem oder mehreren Adjuvantien assoziiert ist.
  22. Immunogene Zusammensetzung nach Anspruch 20 oder 21, dadurch gekennzeichnet, dass sie ferner pharmazeutisch kompatible Trägerstoffe umfasst, wie Wasser, einen Salzpuffer, Dextrose, Glyzerin, Ethanol oder Mischungen aus diesen.
  23. Immunogene Zusammensetzung nach einem beliebigen der Ansprüche 20 bis 22, dadurch gekennzeichnet, dass die methylierte Form natives HBHA ist.
  24. Immunogene Zusammensetzung nach einem beliebigen der Ansprüche 20 bis 22, dadurch gekennzeichnet, dass die methylierte Form ein rekombinantes Peptid gemäß einem beliebigen der Ansprüche 1 bis 7 ist.
  25. Immunogene Zusammensetzung nach einem der Ansprüche 20 bis 24, formuliert für eine nasale, orale, subkutane, intradermale, intramuskuläre, vaginale, rektale, okuläre oder aurikulare Verabreichung.
  26. Immunogene Zusammensetzung nach Anspruch 25, umfassend Hilfsverbindungen, die aus Netzmitteln, Emulgiermitteln oder Puffermitteln gewählt sind.
  27. Immunogene Zusammensetzung nach einem beliebigen der Ansprüche 20 bis 26, dadurch gekennzeichnet, dass sie 0,1 bis 20 µg gereinigtes HBHA-Protein pro Dosis umfasst.
  28. Immunogene Zusammensetzung nach Anspruch 27, dadurch gekennzeichnet, dass sie 5 pµ gereinigtes HBHA-Protein pro Dosis umfasst.
EP02795382A 2001-11-19 2002-11-18 Methyliertes rekombinantes mykobakterielles antigen, dass einem heparin-bindenden hämagglutinin (hbha) entspricht Revoked EP1446422B1 (de)

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EP10183249A EP2354154A1 (de) 2001-11-19 2002-11-18 Methyliertes rekombinantes mykobakterielles Antigen, das einem Heparin-bindenden Hämagglutinin (HBHA) entspricht

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FR0114953A FR2832410B1 (fr) 2001-11-19 2001-11-19 Antigene mycobacterien recombinant de type hemagglutinine de liaison a l'heparine methylee, procedes de preparation et compositions immunogenes comprenant un tel antigene
FR0114953 2001-11-19
PCT/FR2002/003942 WO2003044048A2 (fr) 2001-11-19 2002-11-18 Antigene mycobacterien recombinant de type hemagglutinine de liaison a l'heparine (hbha) methylee

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EP1446422B1 true EP1446422B1 (de) 2011-08-31

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EP02795382A Revoked EP1446422B1 (de) 2001-11-19 2002-11-18 Methyliertes rekombinantes mykobakterielles antigen, dass einem heparin-bindenden hämagglutinin (hbha) entspricht

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FR2872579B1 (fr) * 2004-06-30 2006-11-24 Pasteur Institut Detection de la tuberculose et de l'infection par mycobacterium tuberculosis a l'aide de hbha
CN101370518A (zh) * 2005-01-28 2009-02-18 盖伦生物公司 免疫活性组合物
WO2010149657A1 (en) 2009-06-22 2010-12-29 Px Therapeutics Method for the purification of hbha
CN101816783B (zh) * 2010-04-15 2013-02-06 中国人民解放军第四军医大学 一种表达hbha-il-12融合蛋白的重组耻垢分枝杆菌疫苗
WO2012038486A1 (en) * 2010-09-21 2012-03-29 Inserm (Institut National De La Sante Et De La Recherche Medicale) Method for obtaining a purified hbha polypeptide or fragment thereof
WO2012124641A1 (ja) 2011-03-11 2012-09-20 学校法人東京理科大学 抗癌剤の活性増強剤
GB201116248D0 (en) 2011-09-20 2011-11-02 Glaxosmithkline Biolog Sa Liposome production using isopropanol
WO2013158061A1 (en) * 2012-04-16 2013-10-24 Aeras Global Tb Vaccine Foundation Recombinant mycobacterium encoding a heparin-binding hemagglutinin (hbha) fusion protein and uses thereof
CN110035770B (zh) 2016-12-07 2023-06-16 葛兰素史密丝克莱恩生物有限公司 新方法
GB201621686D0 (en) 2016-12-20 2017-02-01 Glaxosmithkline Biologicals Sa Novel methods for inducing an immune response
GB201707700D0 (en) 2017-05-12 2017-06-28 Glaxosmithkline Biologicals Sa Dried composition
IE87413B1 (en) 2017-05-30 2023-07-19 Glaxosmithkline Biologicals Sa Novel methods for manufacturing an adjuvant
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US11591375B2 (en) 2018-01-22 2023-02-28 Oregon State University Immunogenic compositions comprising Mycobacterium bovis surface proteins and uses thereof
JP2022535091A (ja) 2019-06-05 2022-08-04 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム サポニン精製
EP3757161A1 (de) 2019-06-27 2020-12-30 Schill + Seilacher "Struktol" GmbH Kautschukzusammensetzungen mit polyorganosiloxanen als weichmacher
CN112746051A (zh) * 2020-12-24 2021-05-04 中国人民解放军空军特色医学中心 一种表达甲基化hbha蛋白的重组耻垢分枝杆菌菌株、制备方法及其应用

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FR2793492A1 (fr) * 1999-05-11 2000-11-17 Pasteur Institut Nouveau procede de purification par affinite

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US7829103B2 (en) 2010-11-09
FR2832410A1 (fr) 2003-05-23
JP2010043087A (ja) 2010-02-25
DK1446422T3 (da) 2011-11-14
CA2466937C (fr) 2014-09-02
ES2371597T3 (es) 2012-01-05
US20060292168A1 (en) 2006-12-28
EP1446422A2 (de) 2004-08-18
JP2005526006A (ja) 2005-09-02
AU2002360178A1 (en) 2003-06-10
US8303963B2 (en) 2012-11-06
WO2003044048A3 (fr) 2003-12-11
WO2003044048A2 (fr) 2003-05-30
EP2354154A1 (de) 2011-08-10
ATE522542T1 (de) 2011-09-15
PT1446422E (pt) 2011-11-15
AU2002360178B2 (en) 2008-04-17
FR2832410B1 (fr) 2004-04-02
US20120034257A1 (en) 2012-02-09
CA2466937A1 (fr) 2003-05-30

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